Protein Folding in the Cell – 3

7/30/2019 Protein Folding in the Cell 3

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Protein Folding in the Cell 3

BIOC 212

Winter 2013

J ason C. Young


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HSP70 Cycle

HSP70-ATP HSP70-ADP

Substrate Binding:

HSP70no yes

DNAJ yesno

(transfer to HSP70)

Co-chaperoneinteraction with HSP70

DNAJ NEF


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Chaperonins (HSP60 family)

Chaperonins are large protein complexes with multiple subunits

Typical double-ring structure

E. coli GroEL: 2 rings x 7 subunits x 60 kDa = 840 kDa

In some cases with cap co-chaperone

E. coli GroES cap: 7 subunits x 10 kDa

Act by enclosing substrates within cavity

GroEL

GroES


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GroEL Subunits

Each GroEL subunit has an ATPase domain and an Apical domain Base of the ATPase domain is interface with opposite ring

Movement of the Apical domain is controlled by nucleotide in thesame ring (top) and opposite ring (bottom)

Apical domain down Apical domain up

Apical domain

ATPasedomain

opposite ring

interface


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GroEL Apical Domains

Down position (no nucleotide): hydrophobic surface on apical domains is exposed

provide multiple binding sites for substrate

Up position (ATP and ADP):

hydrophobic surface now binds GroES cap cavity formed with polarsurface

substrate can be encapsulated in cavity and folded

down position no nucleotidehydrophobic (yellow) exposed

can bind polypeptide

up position ATP/ADP

cavity with polar (blue) surfacepolypeptide enclosed

apical domain

GroES

apical domain

cavity


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GroEL Cycle

binds substrate with hydrophobic domains (no nucleotide top ring)

ATP binding (top ring) encloses substrate inside a polar cavity underGroES cap substrate is not bound but is free inside cavity

confinement promotes folding

Substrate is enclosed while ATP is hydrolyzed to ADP no nucleotide (top ring) and ATP (bottom ring) release GroES and

substrate

multiple cycles

Hartl & Hayer-Hartl (2009) NatureStruc Mol Biol 16, 574-581


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HSP70 and Chaperonins

human Hsc70 can act at earlystages when substrate isextended; binds many substrates

human chaperonin TRiC is

specialized for folding certainproteins

TRiC can act after Hsc70, at latestages of folding

no direct contact between Hsc70and TRiC

Young et al. (2004) Nature ReviewsMol. Cell Biol. 5, 781-791.


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HSP90 Family

HSP90 chaperones are homodimers, with 2 identical subunits joinedat the C-termini

human Hsp90: 2 x 90 kDa = 180 kDa

dimer can open and close, like nutcracker

ATP controls opening and closing of the dimer

Ali et al. (2006) Nature 440, 1013-1017

ATP

dimerization

ATP

co-chaperone

p23 (pink)

N middle C


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HSP90 Conformation

Substrate can be bound in nucleotide-free and ATP-bound state

ATP binding allows dimer to close

ATP hydrolysis to ADP compacts the dimer and releases substrate

Shiau et al. (2006) Cell 127, 329340

E. coli Hsp90 (HtpG)

client proteinsbound


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M+Copen position

M+Cclosed position

M+Cwith substrate?

HSP90 Substrate Binding

Thought to bind near-native polypeptides at late stages of folding May bind to hydrophobic surfaces and support a flexible substrate

like a Scaffold

Substrate binding site may be between or alongside subunits

Ali et al. (2006) Nature 440, 1013-1017.

Yeast Hsp90


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HSP90 Proteins

compartment HSP90 co-chaperones

bacterial cytoplasm HtpG no

human cytosolHsp90,

Hsp90yes

human mitochondria Hsp75 no

human ER lumen Grp94 no

* human Hsp90 and are both constitutively expressed and also induced by

stress conditions


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Multichaperone System


Human cytosolic Hsc70 andHsp90 form a multichaperonesystem

Many co-chaperones regulateHsc70 and Hsp90

direct contact to chaperones

many co-chaperones havemodular domains

provide flexibility

folding and non-foldingfunctions


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Hsp90 Co-chaperones

Hsp90 co-chaperones are mostly not essential but increaseefficiency of function

some also interact with Hsc70:

Hop binds Hsc70 and Hsp90 at the same time

ATPaseRegulators

Kinase-specificCo-chaperone

TPR Domain

Co-chaperones

Humans:

Hsp90 p23, Aha1 Cdc37

Hop

FKBP52 (PPIase)

Tom70

many others


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Human Hsp90 ATPase Cycle

Substrate is transferred from Hsc70 to Hsp90 (nucleotide-free)

Hop binds both Hsc70 and Hsp90 to organize transfer Hsp90 in the ATP state binds substrate tightly

co-chaperone p23 stabilizes the closed ATP state of Hsp90

ATP hydrolysis releases substrate from Hsp90


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D, E

Hsc70 and Hsp90 are unrelated but have similar C-terminalsequence motifs:

Hsc70: PTIEEVD-COO- Hsp90: MEEVD-COO-

TPR domains recognize EEVD motifs ionic, hydrogen bonds Hsp70 C-terminal

fragment

Hop TPR1 domain


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TPR Co-chaperones

Hop has 2 TPR domains which bind the Hsc70 and Hsp90 motifs ina similar way, but are still specific for each

modular TPR domains are found in other co-chaperones

recognize Hsc70 or Hsp90 or both

not dependent on nucleotide state of Hsc70 or Hsp90

Hop TPR TPR

Hsc70 Hsp90

CHIP TPR U-box

Hsc70 or Hsp90

ubiquitin ligase

FKBP52 PPI TPR

PPIase

Hsp90 only

PPI


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TPR Domain Co-chaperones

TPR domains are adaptors thatconnect chaperones to differentprotein complexes or locations

FKBP52: PPIase, steroidreceptor chaperone

UNC-45: myosin-specificchaperone

Tom70: mitochondrial import

CHIP: ubiquitin ligase



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Glucocorticoid Receptor

GR belongs to a family of steroid hormone receptors

responds to hormones such as cortisol by activating and repressingspecific genes anti-inflammatory function

ligand binding domain recognizes hormone

DNA binding domain binds to GR promoter elements (GRE) N-terminal activation domain regulates transcription

cortisol

NTD DBD LBD

N-terminal

activation domain

DNA binding

domain

ligand binding

domain

human GR110 kDa


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GR LBD

hydrophobic steroid is bound in the interior of the LBD, and isnecessary to maintain the native structure

in the absence of hormone, LBD cannot fold stably

chaperones keep the LBD partially folded, able to bind hormone

other domains of GR are normally fully folded

partially folded,unstable, inactive

native andactivated

cortisol


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Sequential Action of Chaperones

LBD is folded by a sequence of chaperones

DnaJ A1 (Hsp40) / Hsc70

Hsc70 / Hop / Hsp90

Hsp90 / FKBP52 / p23

without hormone, GR continues to cycle through chaperone system with hormone, GR becomes a stable dimer and active

DnaJ A1: human type 1 Hsp40


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The Heat Shock Response

heat shock followed by recovery period triggers a typical response inhuman cells

translation is inhibited immediately (minutes) and slowly recovers(~1 hour after HS)

transcription of HSPs is up-regulated (6-12 hours); transcription ofother proteins is down-regulated

high expression of chaperones

increased degradation of unfolded proteins

37C

45C

heat shock

recovery

~1 htranslationrecovers

~12 hHSP expression

highest

~24 hreturn tonormal


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HSF1

HSF1 transcription factor is regulator of the heat shock response

DNA binding domain, trimerization domain, and transcriptionactivation domain

Inactive HSF1 is monomeric; active HSF1 is a trimer

Active HSF1 recognizes HSE (heat shock element) promoters


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Regulation of HSF

Voellmy (2004) Cell Stress Chaperones 9, 122-133.

monomeric HSF1 is native, but mimics unfolded protein and isbound by the chaperone Hsp90

after heat shock, Hsp90 binds truly unfolded proteins, and HSF1becomes free to trimerize and activate transcription

chaperones including Hsp90 are expressed and help refold or

degrade misfolded proteins HSF1 is down-regulated by binding of excess Hsp90 to the

monomer form

Hsp90

Inactive HSF

unfoldedproteins

Active Hsfheat

shock


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Compare and Contrast

GR vs. HSF1

chaperone binding, activation, inactivation

Substratebinding:

ATP ADP no nucleotidehow does it

bind substrate

HSP70

Chaperonin

(top ring)

HSP90


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End of 3

Hartl et al. (2011) Molecular chaperones in protein folding andproteostasis. Nature 475, 324-332.

Young et al. (2004) Pathways of chaperone mediated protein folding

in the cytosol. Nature Reviews Mol. Cell Biol. 5, 781-791.

Protein Folding in the Cell – 3

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